Oligonucleotide sequence formula for labeling oligonucleotide probes and proteins for in-situ analysis

ABSTRACT

The present invention provides oligonucleotide probes and oligonucleotide probe collections and protein labeling for detecting or localizing a plurality nucleic acid target genes or antigens within a cell or tissue sample. Specifically, the provides collections of oligonucleotide probe for use in in situ hybridization analyses in which each probe has a label-domain with the sequence formulas of (CTATTTT) n , (AAAATAG) n  or (TTTTATC) n  or (GATAAAA) n  in which all cases “n” would equal 1 or greater. The present invention provides collections or “cocktails” of oligonucleotide probes for detecting or localizing specific nucleic acid target genes within a cell or tissue sample. The cocktails are useful for detecting the following: the Kappa gene (SEQ ID NOS: 1-16 inclusive); the Lamba gene (SEQ ID NOS: 501-509, 511-513, and 515); the CMV (cytomegalovirus) gene (SEQ ID NOS: 221-241 inclusive); EBER (Epstein-Barr RNA) gene (SEQ IN NOS: 51-54 inclusive): Alu (SEQ IN NOS: 55-56); PolyA (SEQ ID NO: 57); and the detection tail (SEQ ID NO: 330).

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to oligonucleotide probes and collectionsof oligonucleotide probes for detecting or localizing nucleic acid genestargets witin a cell or tissue sample. In particular, the inventionrelates to collections of oligoprobes.

[0003] 2. Background of the Invention

[0004] In situ analysis includes in situ hybridization andimmunohistochemistry. In situ hybridization (ISH) employs labeled DNA orRNA probe molecules that are anti-sense to a target gene sequence ortranscript to detect or localize targeted nucleic acid target geneswithin a cell or tissue sample. ISH has proven to be a useful tool in anumber of biomedical fields, including developmental biology, cellbiology, and molecular biology. ISH has been used, for example, todiagnose genetic disorders, map genes, study gene expression, andlocalize sites of target gene expression.

[0005] Typically, ISH is performed by exposing a cell or tissue sampleimmobilized on a glass slide to a labeled nucleic acid probe which iscapable of specifically hybridizing to a given target gene in the cellor tissue sample (In Situ Hybridization: Medical Applications (G. R.Coulton and J. de Belleroche, eds., Kluwer Academic Publishers, 1992);In Situ Hybridization: In Neurobiology; Advances in Methodology (J. H.Eberwine, K. L. Valentino, and J. D. Barchas, eds., Oxford UniversityPress, 1994); In Situ Hybridization: A Practical Approach (D. G.Wilkinson, ed., Oxford University Press, 1992)). The hybridization oflabeled probe molecules to nucleic acids in the cell or tissue samplecan then be detected using, for example, radioactive-based directdetection methods, fluorescence-based direct detection methods, orindirect detection methods based on the binding of afluorescence-labeled protein binding to a hapten such as BrdU,digoxigenin-labeled or biotin-labeled nucleotides incoporated intoprobes. Hapten-based methods have been further extended to include thosemolecules to be bonded by binding protein-enzyme conjugates such asantibody-enzyme-conjugates and colorimetric based detection chemistry.In addition, several target genes can be simulanteously analyzed byexposing a cell or tissue sample to a plurality of nucleic acid probesthat have been labeled with a plurality of different nucleic acid tags.For example, a plurality of nucleic acid probes can be labeled with aplurality of fluorescent compounds having different emissionwavelengths, thereby permitting simultaneous multicolored analysis to beperformed in a single step on a single target cell or tissue sample.

[0006] A significant problem associated with incorporation of labelednucleotides into oligonucleotide probes is that the conjugation moietiesthat are attached to the nucleotide usually interfere with the formationof Watson-Crick base pairing, thus negatively affecting thehybridization of the probe to its target. The has been seen with use oflabel attached via N4-substitued cytosine nucleotides, because of sterichinderance and the expected shift to the less reactive state of asecondary amine (as seen with N4 labled cytosine), as compared to thenatural G—C bond formed with an unsubstituted cytosine (a primaryamine). Any small change or interference with G—C bonding in a smalloligonucleotide (25 to 50 bases) can reduce the ability of these oligosto hybridize with the intended targeted sequence.

[0007] There remains a need in the art to develop suitable probesdesigns for incorporating labeled nucleotides in oligonucleotide probes.We demonstrate that a few artificial seqeunces are viable alternativesfor probe labeling and also work both singly and in complexoligonucleotide probe mixtures for detecting or localizing nucleic acidtarget genes within a cell or tissue sample. The development of suchgeneric seqeunces and labeling strategy for probe collections has wideapplication in the medical, genetic, and molecular biological arts.

[0008] This interference due to labeling chemistry and hybridizationstringency and kinetics is solved herein by designing the oligo to haveat least two distinct functional domains, one domain or sequence to begene specific and involved in the base pair formation, and the seconddomain to be an artificial, non-specific sequence (in reference to thesample's genome) comprised of spacing nucleotides and the labelednucleotide. These elements are positioned so that theselabel-nucleotides are more accessible as haptens for binding proteins(immunoglobulin or avidin(s)) and thus do not interfere withWatson-Crick base pairing in the gene-specific domain.

SUMMARY OF THE INVENTION

[0009] The present invention provides a novel strategy to incorporatelabel into oligonucleotide probes and labeled oligonucleotide probecollections for detecting or localizing nucleic acid target genes withina cell or tissue sample. In particular, the invention relates tonon-gene-specific sequences using sequence formulas for makingrepetitive polymers of such sequences which can be incorporated intocollections of oligonucleotide probes for use in in situ hybridizationanalyses. In addition, using labeled synthetic oligonucleotide polymers,based on sequence formulas, when conjugated to binding proteins, i.e.immunglobulins, is a very effective and controlled process for labelingsuch proteins used in immunohistochemical analysis. The presentinvention provides collections or “cocktails” of oligonucleotide probesfor detecting or localizing specific nucleic acid target genes within acell or tissue sample. The cocktails are useful for detecting thefollowing: the Kappa gene (SEQ ID NOS: 1-16 inclusive); the Lamba gene(SEQ ID NOS: 501-509, 511-513, and 515); the CMV (cytomegalovirus) gene(SEQ ID NOS: 221-241 inclusive); EBER (Epstein-Barr early RNA) gene (SEQID NOS: 51-54 inclusive); Alu (SEQ ID NOS: 55-56); PolyA (SEQ ID NO:57); and the detection tail (SEQ ID NO: 330).

[0010] The invention is directed to an oligonucleotide label-domaincomprising the sequence (CTATTTT)_(n) and its complement (AAAATAG)_(n)wherein “n” is at least 1.

[0011] The invention is also directed to an oligonucleotide probe havingat least two distinct functional domains, a first domain comprising thelabel-domain of claim 2, and a second domain comprising a gene-specifictarget sequence.

[0012] The invention is also directed to a probeset for detecting Kappaimmunoglobulin light chain mRNA or corresponding hetereonuclear RNAwherein the probes are selected from the group consisting essentially ofSEQ ID NOS: 401 through 416, inclusive.

[0013] The invention is also directed to a probeset for detecting Lambdaimmunoglobulin light chain mRNA or corresponding hetereonuclear RNAwherein the probes are selected from the group consisting essentially ofSEQ ID NOS: 501 through 509, 511-513, and 515.

[0014] The invention is also directed to A probeset for detectingcytomegalovirus (CMV) immediate early RNA and/or corresponding mRNAwherein the probes are selected from the group consisting essentially ofSEQ ID NOS: 221 through 241

[0015] The invention is also directed to a probeset for detectingEpstein Barr virus (FBV) early RNA, RNA 1 and RNA 2, (EBER) wherein theprobes are selected from the group consisting essentially of SEQ ID NOS:51 through 54.

[0016] The invention is also directed to a probeset for detecting HumanAlu repetitive sattelite genomic DNA sequences wherein the probes areselected from the group consisting essentially of SEQ ID NOS: 301 and302.

[0017] Specific preferred embodiments of the present invention willbecome evident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 illustrates a generic probe structure of of the two-domainprobe design. This is the oligonucleotide design used for the probes inthe gene specific cocktails described in the following examples. Eachprobe is composed of two domains: a 5′ labeling domain and a 3′ targetgene target gene-specific domain. The labeling domain consists of thisspecific sequence (CTATTTT)n, wherein each cytosine may be labeled witha fluorophore or a cytosine-hapten conjugate, the hapten beingfluorescein in this embodiment. This illustration specifically showsnucleic acid sequences for the 301 (SEQ ID NO: 55) and 302 (SEQ ID NO:56) probes, each of which possesses target gene gene-specific domainscorresponding to human repetitive Alu sequences and labeling domainshaving a fluorescein hapten.

[0019]FIG. 2 illustrates the results obtained for in situ hybridization(ISH) analysis of human skin tissue using a probe comprising thelabeling domain (330 probe; SEQ ID NO: 58). The absence of a detectablesignal indicates that the sequence formula, (CTATTTT)n, of the labelingdomain common to the oligonucleotides used in these ISH examples isnon-specific, and non-reactive in its ability to form Watson-Crick basepairing with human nucleic acid sequences because it does not hybridize.

[0020]FIG. 3 illustrates the results obtained for ISH analysis of humanskin tissue using a probe comprising the labeling domain and a poly d(T)target gene-specific domain (320 probe; SEQ ID NO: 57). The presence ofa detectable signal localized to the cytoplasm indicates that this probeis capable of specifically hybridizing to polyadenylated region ofmessenger RNA.

[0021] FIGS. 4A-4B illustrate the results obtained for ISH analysis ofhuman skin tissue using the 320 probe, wherein the tissue sample was nottreated with ribonuclease A prior to in situ hybridization (A), or wastreated with ribonuclease A prior to in situ hybridization (B). Thedecrease in detectable signal in (B) indicates that this probespecifically hybridizes to polyadenylated region common to messengerRNA.

[0022] FIGS. 5A-5B illustrate the results obtained for ISH analysis ofhuman spleen tissue using the 320 probe, wherein the hybridization andstringency wash were performed at room temperature (A), or at 37° C.(B). This result illustrates that the intenstity of color is related tothe stringency of hybridization conditions, with the more intense colorindicating less stringent conditons.

[0023]FIG. 6 illustrates the results obtained for ISH analysis of thehuman Raji cell line using the 320 probe. This shows that this probedesign also is functional with embedded cell lines as well as embeddedtissue.

[0024]FIG. 7 illustrates the results obtained for ISH analysis of thehuman Raji cell line using a probe collection consisting of the 301 and302 probes.

[0025]FIG. 8 illustrates the results obtained for ISH analysis of thehuman HT cell line using a probe collection consisting of the 301 and302 probes.

[0026]FIG. 9 illustrates the results obtained for ISH analysis of a ratcell line using a probe collection consisting of the 301 and 302 probes.The absence of a detectable signal indicates that this probe collectionis specific for human nucleic acid sequences.

[0027]FIG. 10 illustrates the results obtained for ISH analysis of anEpstein-Barr virus (EBV)-negative human HT cell line using a probepossessing a target gene-specific domain corresponding to EBV EBERnuclear RNA [SEQ ID NO: 51 through SEQ ID NO: 54?].

[0028]FIG. 11 illustrates the results obtained for ISH analysis of humanspleen tissue using a probe collection consisting of probes possessingtarget gene-specific domains corresponding to EBV EBER 1 and 2 nuclearRNA [SEQ ID NO: 51 through SEQ ID NO: 54].

[0029]FIG. 12 illustrates the results obtained for ISH analysis of humantonsil tissue using a probe collection consisting of probes possessingtarget gene-specific domains corresponding to EBV EBER 1 and 2 nuclearRNA [SEQ ID NO: 51 through SEQ ID NO: 54].

[0030] FIGS. 13A-13B illustrate the results obtained for ISH analysis ofhuman spleen tissue using a probe collection consisting of probespossessing target gene-specific domains corresponding to EBV EBER 1 and2 nuclear RNA [SEQ ID NO: 51 through SEQ ID NO: 54, wherein the tissuesample was not treated with ribonuclease A prior to in situhybridization (A), or was treated with ribonuclease A prior to in situhybridization (B). The decrease in detectable signal in (B) indicatesthat this probe specifically hybridizes to human EBER 1 and EBER 2nuclear RNA.

[0031]FIG. 14 illustrates the results obtained for ISH analysis of kappalight chain-positive human tonsil tissue using a probe possessing atarget gene-specific domain corresponding to human immunoglobulin lambdalight chain mRNA [SEQ ID NO: 15].

[0032]FIG. 15 illustrates the results obtained for ISH analysis oflymphoma tissues using a probe collection consisting of probespossessing target-gene-specific domains corresponding tohuman-immunoglobulin kappa light chain mRNA [SEQ ID NOS: 2-4, SEQ IDNOS: 7-12, SEQ ID NOS: 14, 15]. The lymphoma tissue in (A) overexpresses the kappa light chain and the tissue in (B) over expresses thelambda light chain. The absence of a detectable signal in (13) indicatesthat the kappa light chain probe collection is specific to kappa lightchain mRNA.

[0033]FIG. 16 illustrates the results obtained for ISH analysis oflambda light chain-positive human tonsil tissue using a probe possessinga target gene-specific domain corresponding to human inmmunoglobulinlambda light chain variable region mRNA [SEQ ID NOS: 19 through 29].

[0034]FIG. 17 illustrates the results obtained for ISH analysis of alambda light chain-positive human RPMI 8226 cell line using a probecollection consisting of probes possessing target gene-specific domainscorresponding to human immunoglobulin lambda light chain mRNA [SEQ IDNOS: 19 through 29].

[0035] FIGS. 18A-18B illustrate the results obtained for ISH analysis ofhuman spleen tissue using a probe collection consisting of probespossessing target gene-specific domains corresponding to humanimmunoglobulin lambda light chain mRNA [SEQ ID NOS: 19 through 29]. Thetissue in (A) over expresses the lambda light chain and the tissue in(B) over expresses the kappa light chain. The absence of a detectablesignal in (B) indicates that the lambda light chain probe collection isspecific to human lambda light chain mRNA.

[0036]FIG. 19 illustrates the results obtained for ISH analysis ofcytomeglovirus (CMV)-positive human lung tissue using a probe collectionconsisting of probes possessing target gene-specific domainscorresponding to CMV immediate early RNA [SEQ ID NOS: 30-32, SEQ ID NOS:34-35, SEQ ID NO: 38 SEQ ID NO: 50]. [CMV infected cell]

[0037]FIG. 20 illustrates the results obtained for ISH analysis of a rat9G cell line in which the expression of CMV immediate early RNA has notbeen induced by cyclohexamide using a probe collection consisting ofprobes possessing target gene-specific domains corresponding to CMVimmediate early mRNA [SEQ ID NOS: 30-32, SEQ ID NOS: 34-35, SEQ ID NO:38, SEQ ID NO: 50].

[0038] FIGS. 21A-21B illustrate the results obtained for ISH analysis ofa rat 9G cell line in which the expression of CMV immediate early RNAhas been induced by cyclohexamide using a probe collection consisting ofprobes possessing target gene-specific domains corresponding to CMVimmediate early RNA [SEQ ID NOS: 30-32, SEQ ID NOS: 34-35, SEQ ID NO:38, SEQ ID NO: 50. The tissue in (A) is shown at a magnification of 40×and the tissue in (B) is shown at a magnification of 20×.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention provides oligonucleotide probes andoligonucleotide probe collections for detecting or localizing nucleicacid target genes within a cell or tissue sample. In particular, theinvention relates to collections of oligonucleotide probes for use in insitu hybridization analyses.

[0040] More specifically, this invention relates to the use of specificsequence formulas for nucleotide polymers or label-domains to attach adetectable moity (a label) to oligonucleotide probes or proteins. Thespecific utility of these seqeuces or derivatives thereof, is the inertor non-reactive characteristic that does not hybridize to human DNA orRNA at a detectable level under standard stringency of hybridizationconditions. These label-domains or polymers were demonstrated to beuseful generic sequences for incorporation into oligonucleotide probesfor detecting gene-specific sequences within cells or tissue samples inin situ hybridizaiton analyses. Additionally, this inert set ofsequences are useful for attaching a label to inmmunoglobulin or otherproteins for detecting haptens and antigens in immunohistochemicalanalyses.

[0041] As used herein, the terms “probe” or “oligonucleotide probe”refers to a nucleic acid molecule used to detect a complementary nucleicacid target gene.

[0042] As used herein, the term “hybridization” refers to the processwhereby complementary nucleic acid sequences join to form adouble-stranded nucleic acid molecule. By labeling the target nucleicacid molecule with, for example, a radioactive or fluorescent tag,interactions between probe and target genes can be detected.

[0043] The oligonucleotide probes and oligonucleotide probes of thecollections of the present invention are synthesized using conventionalmethods. See e.g., Methods in Molecular Biology, Vol 20: Protocols forOligonucleotides and Analogs 165-89 (S. Agrawal, ed., 1993);Oligonucleotides and Analogues: A Practical Approach 87-108 (F.Eckstein, ed., 1991).

[0044] In a preferred embodiment of the present invention,oligonucleotide probes possess two distinct domains: a 5′ (or labeling)domain and a 3′ (or gene-specific target) domain (See FIG. 1A). In morepreferred embodiments of the present invention, the oligonucleotideprobe possesses a labeling domain which consists of the sequence(CTATTTT)n. Other embodiments are also demonstrated herein, including atriple-domain embodiment having two terminal labeling domains, and acentral gene-specific target domain. Specifically, SEQ ID NOS: 125-126depict this labeling scheme. Yet a further preferred embodiment of alabeling domain is TC(TTTTATC)_(n) or its complement. This sequence ispredicted to be as unique as the (CTATTTT)n label-domain. Theoligonucleotide probes of the present invention are labeled so thathybridization between said probes and target nucleic acids in aparticular cell or tissue can be detected. Labels that are acceptablefor use in in situ hybridization (ISH) analysis are known to those withskill in the art. Such labels permit interactions between probe andtarget genes to be detected using, for example, radioactive-based directdetection methods, fluorescence-based direct detection methods,digoxigenin-labeled or biotin-labeled probes coupled withfluorescence-based detection methods, or digoxigenin-labeled orbiotin-labeled probes coupled with antibody-enzyme-based detectionmethods. In perferred embodiments of the present invention,oligonucleotide probes are labeled with fluorescein. In more preferredembodiments of the present invention, the oligonucleotide probepossesses a labeling domain which consists of the sequence (CTATTTT)n,wherein the cytosine nucleotides may be labeled with a flurophore fordirect detection, or a hapten for indirect detection. In either, thefluorescein-cytosine nucleotide conjugate and the fluorescein moleculeis linked at the N4 position of cytosine through an OBEA linkage (SeeMishra et al., U.S. Pat. No. 5,684,142, which is incorporated herein byreference). In a preferred embodiment, the density of fluorphoreattached to the label-domain is at least 7 mole percent, preferably atleast 10 mole percent, and most preferably at least 16 mole percent,when measured aginst the label-domain solely. For example, if probe 401is considered (a 2-domain probe) it comprises a label-domain of 30 basesincluding a 3′ terminal CT wherein the C is also labeled, the molepercent is 5/30=16.7 mole percent label. In the overall probe, the molepercent is 8.3.

[0045] In some embodiments of the present invention, several targetgenes are simulanteously analyzed by exposing a cell or tissue sample toa plurality of nucleic acid probes that have been labeled with aplurality of different nucleic acid tags. For example, a plurality ofnucleic acid probes can be labeled with a plurality of fluorescentcompounds having different emission wavelengths, thereby permittingsimultaneous multicolored analysis to be performed in a single step on asingle target cell or tissue sample.

[0046] The oligonucleotide probes and oligonucleotide probe collectionsof the present invention may be used in ISH analysis to detect orlocalize nucleic acid target genes within a cell or tissue sample. ISHmay be performed as described, for example, in In Situ Hybridization:Medical Applications (G. R. Coulton and J. de Belleroche, eds., KluwerAcademic Publishers, 1992); In Situ Hybridization: In Neurobiology;Advances in Methodology (J. H. Eberwine, K. L. Valentino, and J. D.Barchas, eds., Oxford University Press, 1994); or In Situ Hybridization:A Practical Approach (D. G. Wilkinson, ed., Oxford University Press,1992)).

[0047] The preferred embodiment of the probes and probe collections ofthe present invention are best understood by referring to FIGS. 1-21 andExamples 1-2. The Examples, which follow, are illustrative of specificembodiments of the invention, and various uses thereof. They are setforth for explanatory purposes only, and are not to be taken as limitingthe invention.

EXAMPLE 1 Probe Collection Preparation

[0048] Probe collections consisting of a plurality of oligonucleotideprobes of 55 to 60 bases in length were designed as follows. In thisExample, each oligonucleotide probe possessed two distinct domains: a 5′(or labeling) domain and a 3′ (or target gene-specific) domain (See FIG.1).

[0049] In this embodiment, the labeling domain consists of the sequence(CTATTTT)n, wherein the cytosine nucleotide represents afluorescein-cytosine nucleotide conjugate and the fluorescein moleculeis linked at the N4 position of cytosine through an OBEA linkage.

[0050] The target gene-specific domain consists of a 25-30 base sequencethat is complementary to a specific nucleic acid target gene.Oligonucleotide probes were designed to possess target gene-specificdomains corresponding to the human immunoglobulin kappa light chainvariable region (See Table 1; oligonucleotide probes 401-416), the humanimmunoglobulin lambda light chain variable region (oligonucleotideprobes 501-515), human cytomegalovirus (CMV) sequences (oligonucleotideprobes 221-241), human Esptein-Barr virus (EBV) EBER (Epstein-Barr earlyRNA) sequences (oligonucleotide probes 100A2, 100C2, 100A1, and 100B1),human repetitive Alu sequences (oligonucleotide probes 301 and 302), andpoly d(T) (oligonucleotide probe 320).

EXAMPLE 2 Label-domain Design: Alu Repetitive Sequence Probe

[0051] Four probes all against the Alu human repetative sequence wereused to evaluate label-domain design. The probes numbered 301 (SEQ IDNO: 55), 301A (SEQ ID NO: 116), 301A2/2 (SEQ ID NO: 121), and 301A3/2(SEQ ID NO: 122) are shown in Table 1.

[0052] The four probes were evaluated at the concentrations of 100, 75,50, and 25 ng/ml per mL of probe in the reaction, respectively. Thishybridization analysis was done manually, using standard protocols. Thetarget, paraffin-embedded cell line MBA MD 468 (Oncor INFORM™ Her-2/neuControl Slides, Cat. No. S8100, Level 1, available from Ventana MedicalSystems, Inc., Tucson, Ariz.) was the target sample and was processed byremoving paraffin by standard xylene methods. The tissue was subjectedto Ventana's. Protease 1 for 12 minutes at 50 degrees C as a 1:2dilution with Ventana's APK buffer. The hybridization reaction wasaccomplished with the addition of probe diluent as 100 ul probe (25%formamide, 5% dextran sulfate, 2×SSC, 1% Triton) to a residual 100 ulvolume of 2×SSC/Triton X-100. The slide was heated to 85 degrees C for 5minutes and then incubated for 1 hr 3? degrees C. Standard SSC washesfollowed for removing excess probe. The hybrids were detected with anantibody against FITC. The mouse antibody was detected colormetricallyusing Ventana Enhanced Alkaline Phosphatase Blue Detection (cat#760-061). Unless otherwise indicated, all reagents were obtained fromVentana Medical Systems, Inc., Tucson, Ariz. The results were observedby colormetric detection using brightfield microscopy.

[0053] The results of these experiments were that signal intensity was afinction of the total number of fluorescein hapten conjugated to theprobe and signal was of the specific label-domain design. The greaterthe number of fluoresceins per probe molecule, the greater the signalobserved. Comparison of design and placement of haptens on the probeshowed that this was not a factor in signal intensity. The two probesthat contained five fluoresceins, (301A3/2 (SEQ ID NO: 122) and 301 (SEQID NO: 55) both yielded equivalent signal. These two probes yieldedgreater signal that seen for 301A2/2, a probe with a split label-domaindesign with four fluoresceins. The probe 301A2/2 yielded a signalgreater than probe 301A a probe with a single label-domain design at the5′ end and with three fluoresceins.

EXAMPLE 3 Label Domain Design: EBER Probes

[0054] This experiment compared two label-domain designs and sequencesto determine whether greater spacing between the fluorescein haptensimproves the production of signal during probe detection steps during insitu hybridization analysis.

[0055] The tissue used was an EBV-infected human spleen tissue fixed inneutral buffered formalin paraffin embedded section of 4-micronthickness placed on silane plus glass microscope slides. The tissuesections were deparaffinized on a Ventana DISCOVERY™ machine, followedby a 6-min digestion with Ventana's Protease 1, at a temperature of 37C. The probe was dissolved in hybridization buffer diluent at aconcentration of 50 ng/mL as a 100 ul applied to an equal volume of2×SSC/Triton X-100 residual volume left on the slide after prepared bythe Ventana Medical Systems, Inc. automated ISH staining system,Discovery. The probe diluent-mixed with the residual volume on slide for6 min at 37 C, then the solution was heated to 85C and held there for atotal of 10 min. The slide was then taken to a 37C temperature and heldat that temperature for 1 hour. All of these aqueous reactions on theslide were all done under a film of LIQUID COVERSLIP™, to preventevaporative loss of water during processing. Each slide afterhybridization was washed 3 times with 2×SSC/Triton solution, with a 6min incubation between each wash, the slide volume being approximately300 ul (+/−10% vol). The hybrids were detected with an antibody againstFITC. The mouse antibody was detected colormetrically using VentanaEnhanced Alkaline Phosphatase Blue Detection (cat# 760-061).

[0056] The two oligonucleotide probes used for this study probe 100A1(SEQ ID NO: 53) and 1002A32 (SEQ ID NO: 120). The two differencesbetween these probes were the label-domain seqence and structure. Theprobe 100A1 label domain was 5′ to gene target domain, contained 5fluoresceins attached to cytosine residues via the OBEA linker, with thesequence formula of (CTATTTT)₄CT (SEQ ID NO: 58). The label domain ofthe oligo probe 1002A32, was similiar, (SEQ ID NO: 125). Besides thedifferent sequence the primary difference was that the fluoresceinelabeled cytosines were spaced 10 bases apart compared to the oligo 100A1the cytosine spacing was closer at 7 bases apart. The result of thiscomparison as deduced by H score analysis were that theseoligonucleotide were equivalent as to the amount of signal generated onthe slide. The data was that for 100A2, for the 368 cells analysed in atotal of 3 fields the H score was 106, and for probe 1002A32 for the 345cell analysed in three field the H score was 109. The H score is aspectrographic analysis done with micrscope that factors into the scorebackground to signal ratio on the tissue section to yield a relativecomparison of total target specific signal on the slide. (See referenceGiroud, F. Perrin C, and Simony Lafontaine, J.; QuantitativeImmunocytochemistry and Immunohistochemistry. Third Conference of theEuropean Society for Analytical Cellular Pathology, 1994; and AutoCyteQuic Immuno User's Manual, 1998, document number PA-029, Co AutoCyteInc. Burlington N.C. 2721). The histograms and the score sheet indicatedthat each oligo were equally efficient in yielding a colormetric signal.This indicates that the position of the label domain can be either 3prime or 5 prime to the gene target sequence or the gene target sequencecan be positioned between two label domains.

EXAMPLE 4 In Situ Hybridization

[0057] The probe collections prepared in Example 1 were first diluted ina solution consisting of 20% dextran sulfate (wt/vol), 50% formamide(vol/vol), 2×SSC, 10 mM Tris-HCl, 5 mM EDTA, and 0.05% Brij-35, at afinal pH of 7.3. Probe collections were then mixed with an equal volumeof a solution consisting of 2×SSC and 0.05% Triton X-100.

[0058] Samples for ISH analysis were prepared by cutting formalin-fixedand paraffin-embedded cells or tissue samples into 4 μm sections andplacing the sections onto a glass slide. Subsequent processing and ISHof samples was carried out in an automated device, such as theDISCOVERY™ Automated ISH/IHC Stainer (Ventana Medical Systems, Inc.,Tucson, Ariz.) described in co-owned and co-pending U.S. Pat.application Ser. Nos. 60/076,198 and 09/259,240, both incorporatedherein by reference. To remove paraffin from the samples, the slideswere immersed in an aqueous solution, heated for approximately 20minutes, and then rinsed. The automated deparaffinization procedure ismore fully described in U.S. Ser. No.60/099,018, 09/259,240 bothincorporated herein by reference. The samples were then treated withprotease and the slides were heated to 85° C. (for hybridization to RNAtarget genes) or 90-95° C. (for hybridization to DNA target genes) for 4to 10 minutes.

[0059] Hybridization reactions were typically performed in ahybridization buffer consisting of 10% dextran sulfate (wt/vol), 25%formamide (vol/vol), 2×SSC, 5 mM Tris, 2.5 mM EDTA, 0.025% Brij-35,0.25% Triton X-100, and between 25 to 125 ng/mL of each individual probemolecule. ISH reactions were performed at between 37° C. to 54° C. ForISH using the probe collections described in Example 1, hybridizationreactions were optimally carried out for 1 hr at 47° C. (except for thepoly d(T) probe, wherein the hybridization reaction was optimallycarried out at 37° C. for 1 hr).

[0060] The hybridization of fluorescein-labeled probe molecules to aparticular target gene in the sample was detected by using a sequentialseries of binding proteins, i.e., secondary antibody detection. However,it is equally possible to use detect detection when visualizing thebound probes. In secondary detection, first, an anti-fluorescein mousemonoclonal antibody directed against the fluorescein-labeled probemolecule was added to the sample. Next, a biotin-labeled polyclonal goatantibody directed against the mouse antibody was added to the sample.Finally, hybridization reactions were colormetrically detected using a5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT)substrate This technigue, termed “secondary antibody detection,” isroutine for one of skill in the art. Primary and secondary antibodiesare available from numerous suppliers, including Ventana MedicalSystems, Tucson, Ariz., which are optimized for use on the Ventanaautostaining systems (ES®, NexES®, DISCOVERY®, and BENCHMARK®).

[0061] FIGS. 2-21 illustrate the results obtained for in situhybridization analysis of various cell lines or tissue samples using theprobes disclosed and claimed herein having the structural motifillustrated in FIG. 1 or probe collections consisting of such probes.

[0062]FIG. 1 illustrates a generic probe structure of of the two-domainprobe design. This is the oligonucleotide design used for the probes inthe gene specific cocktails described in the following examples. Eachprobe is composed of two domains: a 5′ labeling domain and a 3′ targetgene target gene-specific domain. The labeling domain consists of thisspecific sequence (CTATTTT)n, wherein the cytosine nucleotide is acytosine-hapten conjugate, the hapten being fluorescein in thisembodiment. This illustration specifically shows nucleic acid sequencesfor the 301 (SEQ ID NO: 55) and 302 (SEQ ID NO: 56) probes, each ofwhich possesses target gene gene-specific domains corresponding to humanrepetitive Alu sequences and labeling domains having a fluoresceinhapten.

[0063]FIG. 2 illustrates the results obtained for in situ hybridization(ISH) analysis of human skin tissue using a probe comprising thelabeling domain (330 probe; SEQ ID NO: 58). The absence of a detectablesignal indicates that the sequence formula, (CTATTTT)n, of the labelingdomain common to the oligonucleotides used in these ISH examples isnon-specific, and non-reactive in its ability to form Watson-Crick basepairing with human nucleic acid sequences because it does not hybridize.

[0064]FIG. 3 illustrates the results obtained for ISH analysis of humanskin tissue using a probe comprising the labeling domain and a poly d(T)target gene-specific domain (320 probe; SEQ ID NO: 57). The presence ofa detectable signal localized to the cytoplasm indicates that this probeis capable of specifically hybridizing to polyadenylated region ofmessenger RNA.

[0065] FIGS. 4A-4B illustrate the results obtained for ISH analysis ofhuman skin tissue using the 320 probe, wherein the tissue sample was nottreated with ribonuclease A prior to in situ hybridization (A), or wastreated with ribonuclease A prior to in situ hybridization (B). Thedecrease in detectable signal in (B) indicates that this probespecifically hybridizes to polyadenylated region common to messengerRNA.

[0066] FIGS. 5A-5B illustrate the results obtained for ISH analysis ofhuman spleen tissue using the 320 probe, wherein the hybridization andstringency wash were performed at room temperature (A), or at 37° C.(B). This result illustrates that the intenstity of color is related tothe stringency of hybridization conditions, with the more intense colorindicating less stringent conditons.

[0067]FIG. 6 illustrates the results obtained for ISH analysis of thehuman Raji cell line using the 320 probe. This shows that this probedesign also is functional with embedded cell lines as well as embeddedtissue.

[0068]FIG. 7 illustrates the results obtained for ISH analysis of thehuman Raji cell line using a probe collection consisting of the 301 and302 probes.

[0069]FIG. 8 illustrates the results obtained for ISH analysis of thehuman HT cell line using a probe collection consisting of the 301 and302 probes.

[0070]FIG. 9 illustrates the results obtained for ISH analysis of a ratcell line using a probe collection consisting of the 301 and 302 probes.The absence of a detectable signal indicates that this probe collectionis specific for human nucleic acid sequences.

[0071]FIG. 10 illustrates the results obtained for ISH analysis of anEpstein-Barr virus (EBV)-negative human HT cell line using a probepossessing a target gene-specific domain corresponding to EBV EBERnuclear RNA [SEQ ID NO: 51 through SEQ ID NO: 54].

[0072]FIG. 11 illustrates the results obtained for ISH analysis of humanspleen tissue using a probe collection consisting of probes possessingtarget gene-specific domains corresponding to EBV EBER 1 and 2 nuclearRNA [SEQ ID NO: 51 through SEQ ID NO: 54].

[0073]FIG. 12 illustrates the results obtained for ISH analysis of humantonsil tissue using a probe collection consisting of probes possessingtarget gene-specific domains corresponding to EBV EBER 1 and 2 nuclearRNA [SEQ ID NO: 51 through SEQ ID NO: 54].

[0074] FIGS. 13A-13B illustrate the results obtained for ISH analysis ofhuman spleen tissue using a probe collection consisting of probespossessing target gene-specific domains corresponding to EBV EBER 1 and2 nuclear RNA [SEQ ID NO: 51 through SEQ ID NO: 54], wherein the tissuesample was not treated with ribonuclease A prior to in situhybridization (A), or was treated with ribonuclease A prior to in situhybridization (B). The decrease in detectable signal in (B) indicatesthat this probe specifically hybridizes to human EBER 1 and EBER 2nuclear RNA.

[0075]FIG. 14 illustrates the results obtained for ISH analysis of kappalight chain-positive human tonsil tissue using a probe possessing atarget gene-specific domain corresponding to human immunoglobulin lambdalight chain mRNA [SEQ ID NO: 15].

[0076]FIG. 15 illustrates the results obtained for ISH analysis oflymphoma tissues using a probe collection consisting of probespossessing target gene-specific domains corresponding to humanimmunoglobulin kappa light chain mRNA [SEQ ID NOS: 2-4, SEQ ID NOS:7-12, SEQ ID NOS: 14, 15]. The lymphoma tissue in (A) over expresses thekappa light chain and the tissue in (B) over expresses the lambda lightchain. The absence of a detectable signal in (B) indicates that thekappa light chain probe collection is specific to kappa light chainmRNA.

[0077]FIG. 16 illustrates the results obtained for ISH analysis oflambda light chain-positive human tonsil tissue using a probe possessinga target gene-specific domain corresponding to human immunoglobulinlambda light chain variable region mRNA [SEQ ID NOS: 19 through 29].

[0078]FIG. 17 illustrates the results obtained for ISH analysis of alambda light chain-positive human RPMI 8226 cell line using a probecollection consisting of probes possessing target gene-specific domainscorresponding to human immunoglobulin lambda light chain mRNA [SEQ IDNOS: 19 through 29].

[0079] FIGS. 18A-18B illustrate the results obtained for ISH analysis ofhuman spleen tissue using a probe collection consisting of probespossessing target gene-specific domains corresponding to humanimmunoglobulin lambda light chain mRNA [SEQ ID NOS: 19 through 29]. Thetissue in (A) over expresses the lambda light chain and the tissue in(B) over expresses the kappa light chain. The absence of a detectablesignal in (B) indicates that the lambda light chain probe collection isspecific to human lambda light chain mRNA.

[0080]FIG. 19 illustrates the results obtained for ISH analysis ofcytomeglovirus (CMV)-positive human lung tissue using a probe collectionconsisting of probes possessing target gene-specific domainscorresponding to CMV immediate early RNA [SEQ ID NOS: 30-32, SEQ ID NOS:34-35, SEQ ID NO: 38, SEQ ID NO: 50]. Arrow indicates CMV infected cell.

[0081]FIG. 20 illustrates the results obtained for ISH analysis of a rat9G cell line in which the expression of CMV immediate early RNA has notbeen induced by cyclohexamide using a probe collection consisting ofprobes possessing target gene-specific domains corresponding to CMVimmediate early RNA [SEQ ID NOS: 30-32, SEQ ID NOS: 34-35, SEQ ID NO:38, SEQ ID NO: 50].

[0082] FIGS. 21A-21B illustrate the results obtained for ISH analysis ofa rat 9G cell line in which the expression of CMV immediate early RNAhas been induced by cyclohexamide using a probe collection consisting ofprobes possessing target gene-specific domains corresponding to CMVimmediate early RNA [SEQ ID NOS: 30-32, SEQ ID NOS: 34-35, SEQ ID NO:38, SEQ ID NO: 50] expression of the CMV immediate early RNA withcyclohexamide. The tissue in (A) is shown at a magnification of 40× andthe tissue in (B) is at a magnification of 20×. TABLE 1 Probe SEQ IDSequence ID 4015′-CTATTTTCTATTTTCTATTTTCTATTTTCT  CCAGAGTAGCAGGAGCCCCAGGAGCTGAGC-3′ 1402 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GGATGGAGACTGGGTCAACTGGATGTCACA-3′2 4035′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GCAAGCGATGGTGACTCTGTCTCCTACAGC-3′ 3404 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TCTGTCCCAGATCCACTGCCACTGAACCTT-3′4 4055′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GCAGCCACAGTTCGCTTCATCTGCACCTTG-3′ 5406 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TTTCAACTGCTCATCAGATGGCGGGAAGAT-3′6 4075′-CTATTTTCTATTTTCTATTTTCTATTTTCT  AAGTTATTCAGCAGGCACACAACAGAGGCA-3′ 7408 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GGCGTTATCCACCTTCCACTGTACTTTGGC-3′8 4095′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TAGGTGCTGTCCTTGCTGTCCTGCTCTGTG-3′ 9410 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GTAGTCTGCTTTGCTCAGCGTCAGGGTGCT-3′10 4115′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GATGGGTGACTTCGCAGGCGTAGACTTTGT-3′ 11412 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  CTCTCCCCTGTTGAAGCTCTTTGTGACGGG-3′12 4135′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TGGAACTGAGGAGCAGGTGGGGGCACTTCT-3′ 13414 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GAAAAAGGGTCAGAGGCCAAAGGATGGGAG-3′14 4155′-CTATTTTCTATTTTCTATTTTCTATTTTCT  AGATGAGCTGGAGGACCGCAATAGGGGTAG-3′ 15416 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GCATAATTAAAGCCAAGGAGGAGGAGGGGG-3′16 5015′-CTATTTTCTATTTTCTATTTTCTATTTTCT  CCTGAGTGAGGAGGGTGAGGAGCAGCAGAG-3′ 17502 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  AGACCCAGACACGGAGGCAGGCTGAGTCAG-3′18 5035′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TGTTGGTTCCAGTGCAGGAGATGGTGATCG-3′ 19504 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TAAATCATGATTTTGGGGGCTTTGCCTGGG-3′20 5055′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TGTTGCCAGACTTGGAGCCAGAGAAGCGAT-3′ 21506 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  AATAATCAGCCTCGTCCTCAGCCTGGAGCC-3′22 5075′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GGTCCCTCCGCCGAAAACCACAGTGTAACT-3′ 23508 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TTATGAGACACACCAGTGTGGCCTTGTTGG-3′24 5095′-CTATTTTCTATTTTCTATTTTCTATTTTCT  CTGCTCAGGCGTCAGGCTCAGATAGCTGCT-3′ 25511 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  ATGCGTGACCTGGCAGCTGTAGCTTCTGTG-3′26 5125′-CTATTTTCTATTTTCTATTTTCTATTTTCT  ATTCTGTAGGGGCCACTGTCTTCTCCACGG-3′ 27513 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  CCTCCCCTGGGATCCTGCAGCTCTAGTCTC-3′28 5155′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TGAGGGTTTATTGAGTGCAGGGAGAAGGGC-3′ 29221 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GGAGGTCAAAACAGCGTGGATGGCG-3′ 30222 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GAGGCTGGATCGGTCCCGGTGTCTT-3′ 31223 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  AATCCGCGTTCCAATGCACCGTTCC-3′ 32224 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TAAAAACTGCGGGCACTGGGGACGG-3′ 33225 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  ACCCGAGATTCGCGTGGAGATCCCA-3′ 34226 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GAGCAAGGAGCTGCCGAGCGACCAT-3′ 35227 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  ACACTGGTGGTGGTGGGCATCGTGC-3′ 36228 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TTCCAAATGCGTCAGCGGTGCAAGC-3′ 37229 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  AGCTGCCTGCATCTTCTTCTGCCGC-3′ 38238 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TCTCAGAGGATCGGCCCCCAGAATG-3′ 47239 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  CCTCATCTGACTCCTCGGCGATGGC-3′ 48240 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  CGGGTACAGGGGACTCTGGGGGTGA-3′ 49241 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GGGTGGGTGCTCTTGCCTCCAGAGG-3′ 50100A2 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GACCTCGGGTCGGTAGCACCGCACT-3′ 51100C2 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GGAAGCCTCTCTTCTCCTCCCCCGG-3′ 52100A1 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  CCACAGACACCGTCCTCACCACCCG-3′ 53100B1 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  GGCTACAGCCACACACGTCTCCTCC-3′ 54301 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  CGAGGCGGGCGGATCACCTGAGGTC-3′ 55302 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  CGGGAGGCGGAGGTTGCAGTGAGCC-3′ 56320 5′-CTATTTTCTATTTTCTATTTTCTATTTTCT  TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′57 301A 5′-CTATTTTTCTATTTTTCTTTT  CGAGGCGGGCGGATCACCTGAGGTC-3′ 116 302C5′-CTATTTTTCTATTTTTCTTTT  CGGGAGGCGGAGGTTGCAGTGAGCC-3′ 117 302A45′-CTATTTTATACTTTATATTTCATATTTTATCT  CGGGAGGCGGAGGTTGCAGTGAGCC-3′ 118302A3/25′-CTATTTTATATTTATATTTCT  CGGGAGGCGGAGGTTGCAGTGAGCC  ACTATTTTATACTT-3119 1002A32 5′-CTATTTTATACTTTATATTTCTGACCTCGGGTCGGTAGCACCGCAC TACTATTTTATACTT-3′ 120 301A2/2 5′-CTATTTTTCTTCGAGGCGGGCGGATCACCTGAGGTC TTCTTTTTATCTT-3 121 301A3/25′-CTATTTTATACTTTATATTTCT CGAGGCGGGCGGATCACCTGAGGTC ACTATTTTATACTT-3′122

[0083] TABLE 2 Probe SEQ ID Sequence ID 5′-CTATTTTTCTATTTTTCTTTT 1235′-CTATTTTATACTTTATATTTCATATTTTATCT 124 3305′-CTATTTTCTATTTTCTATTTTCTATTTTCT 585′-CTATTTTATACTTTATATTTCT...........ACTATTTTATACTT-3 1255′-CTATTTTTCTT...........TTCTTTTTATCTT-3 126

[0084] It should be understood that the foregoing disclosure emphasizescertain specific embodiments of the invention and that all modificationsor alternatives equivalent thereto are within the spirit and scope ofthe invention as set forth in the appended claims.

1 126 1 60 DNA Artificial Sequence oligonucleotide probe 1 ctattttctattttctattt tctattttct ccagagtagc aggagcccca ggagctgagc 60 2 60 DNAArtificial Sequence oligonucleotide probe 2 ctattttcta ttttctattttctattttct ggatggagac tgggtcaact ggatgtcaca 60 3 60 DNA ArtificialSequence oligonucleotide probe 3 ctattttcta ttttctattt tctattttctgcaagcgatg gtgactctgt ctcctacagc 60 4 60 DNA Artificial Sequenceoligonucleotide probe 4 ctattttcta ttttctattt tctattttct tctgtcccagatccactgcc actgaacctt 60 5 60 DNA Artificial Sequence oligonucleotideprobe 5 ctattttcta ttttctattt tctattttct gcagccacag ttcgcttcatctgcaccttg 60 6 60 DNA Artificial Sequence oligonucleotide probe 6ctattttcta ttttctattt tctattttct tttcaactgc tcatcagatg gcgggaagat 60 760 DNA Artificial Sequence oligonucleotide probe 7 ctattttcta ttttctattttctattttct aagttattca gcaggcacac aacagaggca 60 8 60 DNA ArtificialSequence oligonucleotide probe 8 ctattttcta ttttctattt tctattttctggcgttatcc accttccact gtactttggc 60 9 60 DNA Artificial Sequenceoligonucleotide probe 9 ctattttcta ttttctattt tctattttct taggtgctgtccttgctgtc ctgctctgtg 60 10 60 DNA Artificial Sequence oligonucleotideprobe 10 ctattttcta ttttctattt tctattttct gtagtctgct ttgctcagcgtcagggtgct 60 11 60 DNA Artificial Sequence oligonucleotide probe 11ctattttcta ttttctattt tctattttct gatgggtgac ttcgcaggcg tagactttgt 60 1260 DNA Artificial Sequence oligonucleotide probe 12 ctattttctattttctattt tctattttct ctctcccctg ttgaagctct ttgtgacggg 60 13 60 DNAArtificial Sequence oligonucleotide probe 13 ctattttcta ttttctattttctattttct tggaactgag gagcaggtgg gggcacttct 60 14 60 DNA ArtificialSequence oligonucleotide probe 14 ctattttcta ttttctattt tctattttctgaaaaagggt cagaggccaa aggatgggag 60 15 60 DNA Artificial Sequenceoligonucleotide probe 15 ctattttcta ttttctattt tctattttct agatgagctggaggaccgca ataggggtag 60 16 60 DNA Artificial Sequence oligonucleotideprobe 16 ctattttcta ttttctattt tctattttct gcataattaa agccaaggaggaggaggggg 60 17 60 DNA Artificial Sequence oligonucleotide probe 17ctattttcta ttttctattt tctattttct cctgagtgag gagggtgagg agcagcagag 60 1860 DNA Artificial Sequence oligonucleotide probe 18 ctattttctattttctattt tctattttct agacccagac acggaggcag gctgagtcag 60 19 60 DNAArtificial Sequence oligonucleotide probe 19 ctattttcta ttttctattttctattttct tgttggttcc agtgcaggag atggtgatcg 60 20 60 DNA ArtificialSequence oligonucleotide probe 20 ctattttcta ttttctattt tctattttcttaaatcatga ttttgggggc tttgcctggg 60 21 60 DNA Artificial Sequenceoligonucleotide probe 21 ctattttcta ttttctattt tctattttct tgttgccagacttggagcca gagaagcgat 60 22 60 DNA Artificial Sequence oligonucleotideprobe 22 ctattttcta ttttctattt tctattttct aataatcagc ctcgtcctcagcctggagcc 60 23 60 DNA Artificial Sequence oligonucleotide probe 23ctattttcta ttttctattt tctattttct ggtccctccg ccgaaaacca cagtgtaact 60 2460 DNA Artificial Sequence oligonucleotide probe 24 ctattttctattttctattt tctattttct ttatgagaca caccagtgtg gccttgttgg 60 25 60 DNAArtificial Sequence oligonucleotide probe 25 ctattttcta ttttctattttctattttct ctgctcaggc gtcaggctca gatagctgct 60 26 60 DNA ArtificialSequence oligonucleotide probe 26 ctattttcta ttttctattt tctattttctatgcgtgacc tggcagctgt agcttctgtg 60 27 60 DNA Artificial Sequenceoligonucleotide probe 27 ctattttcta ttttctattt tctattttct attctgtaggggccactgtc ttctccacgg 60 28 60 DNA Artificial Sequence oligonucleotideprobe 28 ctattttcta ttttctattt tctattttct cctcccctgg gatcctgcagctctagtctc 60 29 60 DNA Artificial Sequence oligonucleotide probe 29ctattttcta ttttctattt tctattttct tgagggttta ttgagtgcag ggagaagggc 60 3055 DNA Artificial Sequence oligonucleotide probe 30 ctattttctattttctattt tctattttct ggaggtcaaa acagcgtgga tggcg 55 31 55 DNAArtificial Sequence oligonucleotide probe 31 ctattttcta ttttctattttctattttct gaggctggat cggtcccggt gtctt 55 32 55 DNA Artificial Sequenceoligonucleotide probe 32 ctattttcta ttttctattt tctattttct aatccgcgttccaatgcacc gttcc 55 33 55 DNA Artificial Sequence oligonucleotide probe33 ctattttcta ttttctattt tctattttct taaaaactgc gggcactggg gacgg 55 34 55DNA Artificial Sequence oligonucleotide probe 34 ctattttcta ttttctattttctattttct acccgagatt cgcgtggaga tccca 55 35 55 DNA Artificial Sequenceoligonucleotide probe 35 ctattttcta ttttctattt tctattttct gagcaaggagctgccgagcg accat 55 36 55 DNA Artificial Sequence oligonucleotide probe36 ctattttcta ttttctattt tctattttct acactggtgg tggtgggcat cgtgc 55 37 55DNA Artificial Sequence oligonucleotide probe 37 ctattttcta ttttctattttctattttct ttccaaatgc gtcagcggtg caagc 55 38 55 DNA Artificial Sequenceoligonucleotide probe 38 ctattttcta ttttctattt tctattttct agctgcctgcatcttcttct gccgc 55 39 55 DNA Artificial Sequence oligonucleotide probe39 ctattttcta ttttctattt tctattttct ccctccaccg ttaacagcac cgcaa 55 40 55DNA Artificial Sequence oligonucleotide probe 40 ctattttcta ttttctattttctattttct ttggtcacgg gtgtctcggg cctaa 55 41 55 DNA Artificial Sequenceoligonucleotide probe 41 ctattttcta ttttctattt tctattttct tcggccaactctggaaacag cgggt 55 42 55 DNA Artificial Sequence oligonucleotide probe42 ctattttcta ttttctattt tctattttct tcggggttct cgttgcaatc ctcgg 55 43 55DNA Artificial Sequence oligonucleotide probe 43 ctattttcta ttttctattttctattttct atctcgatgc cccgctcaca tgcaa 55 44 55 DNA Artificial Sequenceoligonucleotide probe 44 ctattttcta ttttctattt tctattttct tgccgcaccatgtccactcg aacct 55 45 55 DNA Artificial Sequence oligonucleotide probe45 ctattttcta ttttctattt tctattttct gttagcggcg cccttgctca catca 55 46 55DNA Artificial Sequence oligonucleotide probe 46 ctattttcta ttttctattttctattttct tgcagatctc ctcaatgcgg cgctt 55 47 55 DNA Artificial Sequenceoligonucleotide probe 47 ctattttcta ttttctattt tctattttct tctcagaggatcggccccca gaatg 55 48 55 DNA Artificial Sequence oligonucleotide probe48 ctattttcta ttttctattt tctattttct cctcatctga ctcctcggcg atggc 55 49 55DNA Artificial Sequence oligonucleotide probe 49 ctattttcta ttttctattttctattttct cgggtacagg ggactctggg ggtga 55 50 55 DNA Artificial Sequenceoligonucleotide probe 50 ctattttcta ttttctattt tctattttct gggtgggtgctcttgcctcc agagg 55 51 55 DNA Artificial Sequence oligonucleotide probe51 ctattttcta ttttctattt tctattttct gacctcgggt cggtagcacc gcact 55 52 55DNA Artificial Sequence oligonucleotide probe 52 ctattttcta ttttctattttctattttct ggaagcctct cttctcctcc cccgg 55 53 55 DNA Artificial Sequenceoligonucleotide probe 53 ctattttcta ttttctattt tctattttct ccacagacaccgtcctcacc acccg 55 54 56 DNA Artificial Sequence oligonucleotide probe54 ctattttcta ttttctattt tctattttct ggctacagcc acacacgtct cctccc 56 5555 DNA Artificial Sequence oligonucleotide probe 55 ctattttctattttctattt tctattttct cgaggcgggc ggatcacctg aggtc 55 56 55 DNAArtificial Sequence oligonucleotide probe 56 ctattttcta ttttctattttctattttct cgggaggcgg aggttgcagt gagcc 55 57 60 DNA Artificial Sequenceoligonucleotide probe 57 ctattttcta ttttctattt tctattttct tttttttttttttttttttt tttttttttt 60 58 30 DNA Artificial Sequence oligonucleotideprobe 58 ctattttcta ttttctattt tctattttct 30 59 30 DNA ArtificialSequence oligonucleotide probe 59 ccagagtagc aggagcccca ggagctgagc 30 6030 DNA Artificial Sequence oligonucleotide probe 60 ggatggagactgggtcaact ggatgtcaca 30 61 30 DNA Artificial Sequence oligonucleotideprobe 61 gcaagcgatg gtgactctgt ctcctacagc 30 62 30 DNA ArtificialSequence oligonucleotide probe 62 tctgtcccag atccactgcc actgaacctt 30 6330 DNA Artificial Sequence oligonucleotide probe 63 gcagccacagttcgcttcat ctgcaccttg 30 64 30 DNA Artificial Sequence oligonucleotideprobe 64 tttcaactgc tcatcagatg gcgggaagat 30 65 30 DNA ArtificialSequence oligonucleotide probe 65 aagttattca gcaggcacac aacagaggca 30 6630 DNA Artificial Sequence oligonucleotide probe 66 ggcgttatccaccttccact gtactttggc 30 67 30 DNA Artificial Sequence oligonucleotideprobe 67 taggtgctgt ccttgctgtc ctgctctgtg 30 68 30 DNA ArtificialSequence oligonucleotide probe 68 gtagtctgct ttgctcagcg tcagggtgct 30 6930 DNA Artificial Sequence oligonucleotide probe 69 gatgggtgacttcgcaggcg tagactttgt 30 70 30 DNA Artificial Sequence oligonucleotideprobe 70 ctctcccctg ttgaagctct ttgtgacggg 30 71 30 DNA ArtificialSequence oligonucleotide probe 71 tggaactgag gagcaggtgg gggcacttct 30 7230 DNA Artificial Sequence oligonucleotide probe 72 gaaaaagggtcagaggccaa aggatgggag 30 73 30 DNA Artificial Sequence oligonucleotideprobe 73 agatgagctg gaggaccgca ataggggtag 30 74 30 DNA ArtificialSequence oligonucleotide probe 74 gcataattaa agccaaggag gaggaggggg 30 7530 DNA Artificial Sequence oligonucleotide probe 75 cctgagtgaggagggtgagg agcagcagag 30 76 30 DNA Artificial Sequence oligonucleotideprobe 76 agacccagac acggaggcag gctgagtcag 30 77 30 DNA ArtificialSequence oligonucleotide probe 77 tgttggttcc agtgcaggag atggtgatcg 30 7830 DNA Artificial Sequence oligonucleotide probe 78 taaatcatgattttgggggc tttgcctggg 30 79 30 DNA Artificial Sequence oligonucleotideprobe 79 tgttgccaga cttggagcca gagaagcgat 30 80 30 DNA ArtificialSequence oligonucleotide probe 80 aataatcagc ctcgtcctca gcctggagcc 30 8130 DNA Artificial Sequence oligonucleotide probe 81 ggtccctccgccgaaaacca cagtgtaact 30 82 30 DNA Artificial Sequence oligonucleotideprobe 82 ttatgagaca caccagtgtg gccttgttgg 30 83 30 DNA ArtificialSequence oligonucleotide probe 83 ctgctcaggc gtcaggctca gatagctgct 30 8430 DNA Artificial Sequence oligonucleotide probe 84 atgcgtgacctggcagctgt agcttctgtg 30 85 30 DNA Artificial Sequence oligonucleotideprobe 85 attctgtagg ggccactgtc ttctccacgg 30 86 30 DNA ArtificialSequence oligonucleotide probe 86 cctcccctgg gatcctgcag ctctagtctc 30 8730 DNA Artificial Sequence oligonucleotide probe 87 tgagggtttattgagtgcag ggagaagggc 30 88 25 DNA Artificial Sequence oligonucleotideprobe 88 ggaggtcaaa acagcgtgga tggcg 25 89 25 DNA Artificial Sequenceoligonucleotide probe 89 gaggctggat cggtcccggt gtctt 25 90 25 DNAArtificial Sequence oligonucleotide probe 90 aatccgcgtt ccaatgcacc gttcc25 91 25 DNA Artificial Sequence oligonucleotide probe 91 taaaaactgcgggcactggg gacgg 25 92 25 DNA Artificial Sequence oligonucleotide probe92 acccgagatt cgcgtggaga tccca 25 93 25 DNA Artificial Sequenceoligonucleotide probe 93 gagcaaggag ctgccgagcg accat 25 94 25 DNAArtificial Sequence oligonucleotide probe 94 acactggtgg tggtgggcat cgtgc25 95 25 DNA Artificial Sequence oligonucleotide probe 95 ttccaaatgcgtcagcggtg caagc 25 96 25 DNA Artificial Sequence oligonucleotide probe96 agctgcctgc atcttcttct gccgc 25 97 25 DNA Artificial Sequenceoligonucleotide probe 97 ccctccaccg ttaacagcac cgcaa 25 98 25 DNAArtificial Sequence oligonucleotide probe 98 ttggtcacgg gtgtctcggg cctaa25 99 25 DNA Artificial Sequence oligonucleotide probe 99 tcggccaactctggaaacag cgggt 25 100 25 DNA Artificial Sequence oligonucleotide probe100 tcggggttct cgttgcaatc ctcgg 25 101 25 DNA Artificial Sequenceoligonucleotide probe 101 atctcgatgc cccgctcaca tgcaa 25 102 25 DNAArtificial Sequence oligonucleotide probe 102 tgccgcacca tgtccactcgaacct 25 103 25 DNA Artificial Sequence oligonucleotide probe 103gttagcggcg cccttgctca catca 25 104 25 DNA Artificial Sequenceoligonucleotide probe 104 tgcagatctc ctcaatgcgg cgctt 25 105 25 DNAArtificial Sequence oligonucleotide probe 105 tctcagagga tcggcccccagaatg 25 106 25 DNA Artificial Sequence oligonucleotide probe 106cctcatctga ctcctcggcg atggc 25 107 25 DNA Artificial Sequenceoligonucleotide probe 107 cgggtacagg ggactctggg ggtga 25 108 25 DNAArtificial Sequence oligonucleotide probe 108 gggtgggtgc tcttgcctccagagg 25 109 25 DNA Artificial Sequence oligonucleotide probe 109gacctcgggt cggtagcacc gcact 25 110 25 DNA Artificial Sequenceoligonucleotide probe 110 ggaagcctct cttctcctcc cccgg 25 111 25 DNAArtificial Sequence oligonucleotide probe 111 ccacagacac cgtcctcaccacccg 25 112 26 DNA Artificial Sequence oligonucleotide probe 112ggctacagcc acacacgtct cctccc 26 113 25 DNA Artificial Sequenceoligonucleotide probe 113 cgaggcgggc ggatcacctg aggtc 25 114 25 DNAArtificial Sequence oligonucleotide probe 114 cgggaggcgg aggttgcagtgagcc 25 115 30 DNA Artificial Sequence oligonucleotide probe 115tttttttttt tttttttttt tttttttttt 30 116 46 DNA Artificial Sequenceoligonucleotide probe 116 ctatttttct atttttcttt tcgaggcggg cggatcacctgaggtc 46 117 46 DNA Artificial Sequence oligonucleotide probe 117ctatttttct atttttcttt tcgggaggcg gaggttgcag tgagcc 46 118 57 DNAArtificial Sequence oligonucleotide probe 118 ctattttata ctttatatttcatattttat ctcgggaggc ggaggttgca gtgagcc 57 119 60 DNA ArtificialSequence oligonucleotide probe 119 ctattttata tttatatttc tcgggaggcggaggttgcag tgagccacta ttttatactt 60 120 61 DNA Artificial Sequenceoligonucleotide probe 120 ctattttata ctttatattt ctgacctcgg gtcggtagcaccgcactact attttatact 60 t 61 121 49 DNA Artificial Sequenceoligonucleotide probe 121 ctatttttct tcgaggcggg cggatcacct gaggtcttctttttatctt 49 122 61 DNA Artificial Sequence oligonucleotide probe 122ctattttata ctttatattt ctcgaggcgg gcggatcacc tgaggtcact attttatact 60 t61 123 21 DNA Artificial Sequence oligonucleotide probe 123 ctatttttctatttttcttt t 21 124 32 DNA Artificial Sequence oligonucleotide probe 124ctattttata ctttatattt catattttat ct 32 125 61 DNA Artificial Sequenceoligonucleotide probe 125 ctattttata ctttatattt ctnnnnnnnn nnnnnnnnnnnnnnnnnact attttatact 60 t 61 126 49 DNA Artificial Sequenceoligonucleotide probe 126 ctatttttct tnnnnnnnnn nnnnnnnnnn nnnnnnttctttttatctt 49

We claim:
 1. An oligonucleotide label-domain comprising the sequence(CTATTTT)_(n) and its complement (AAAATAG)_(n) wherein “n” is atleast
 1. 2. The oligonucleotide label-domain of claim 1 detectablylabeled with a reporter molecule, or a hapten molecule.
 3. Theoligonucleotide label-domain of claim 2 wherein the hapten isflurorescein linked to the N4 nitrogen of cytosine through an OBEAlinker.
 4. The oligonucleotide label-domain of claim 1 wherein thereporter molecule is a fluorophore.
 5. The oligonucleotide label-domainof claim 1 wherein the fluorophore is present at a density of greaterthan 7 mole percent.
 6. The oligonucleotide label-domain of claim 1wherein the label-domain has the sequence TC(TTTTATC)_(n) (or itscomplementary formula).
 7. The oligonucleotide label-domain of claim 1wherein the sequence is SEQ ID NO:
 58. 8. The oligonucleotidelabel-domain of claim 2 wherein at least 7 mole pecent of the cytosinesare linked to a detectable moiety by an OBEA linker.
 9. Anoligonucleotide probe having at least two distinct functional domains, afirst domain comprising the label-domain of claim 2, and a second domaincomprising a gene-specific target sequence.
 10. The oligonucleotideprobe of claim 9 wherein the label-domain is located at the 5′ end ofthe oligonucleotide probe, and the gene-specific target sequence being3′ to the label-domain.
 11. The oligonucleotide probe of claim 9 whereinthe label-domain is located at the 3′ end of the oligonucleotide probe,and the gene-specific target sequence is 5′ to the label-domain.
 12. Anoligonucleotide probe having three distinct functional domains, a firstdomain comprising the label-domain of claim 2, a second domaincomprising a gene-specific target sequence, and a third domaincomprising another label-domain, wherein said second domain is locatedbetween said first and third domains.
 13. A probeset for detecting Kappaimmunoglobulin light chain mRNA or corresponding hetereonuclear RNAwherein the probes are selected from the group consisting essentially ofSEQ ID NOS: 401 through 416, inclusive.
 14. A probeset for detectingLambda immunoglobulin light chain mRNA or corresponding hetereonuclearRNA wherein the probes are selected from the group consistingessentially of SEQ ID NOS: 501 through 509, 511-513, and
 515. 15. Aprobeset for detecting cytomegalovirus (CMV) immediate early RNA and/orcorresponding mRNA wherein the probes are selected from the groupconsisting essentially of SEQ ID NOS: 221 through 241
 16. A probeset fordetecting Epstein Barr virus (EBV) early RNA, RNA 1 and RNA 2, (EBER)wherein the probes are selected from the group consisting essentially ofSEQ ID NOS: 51 through
 54. 17. A probeset for detecting Human Alurepetitive sattelite genomic DNA sequences wherein the probes areselected from the group consisting essentially of SEQ ID NOS: 301 and302.